The long-term goal of this project is to discover novel types of broad-spectrum antibiotics capable of effectively eradicating a wide range of potential biowarfare agents, as well as conventional pathogens. In this phase of the project, we will develop a high-throughput screen for a targeted discovery of prodrug antibiotics. The screen is aimed at solving the following problems that have impeded antimicrobial drug discovery: automatically eliminate a high background of generally- toxic molecules in compound library screening; obtain lead compounds with good penetration into Gram negative species, indicative of a broad spectrum of activity; and an ability to sterilize an infection by eliminating persister cells invulnerable to all current antibiotics. Prodrugs such as metronidazole convert inside the cell into a reactive molecule that hits multiple targets. In this regard, an activated prodrug acts like an antiseptic. Our preliminary data show that prodrugs can potentially completely sterilize a stationary state population of E. coli containing persisters. An ability to sterilize an infection can be critically important for treating immunocompromised individuals and multidrug tolerant biofilms that cause 65% of all infectious diseases in developed countries. An activated prodrug will bind covalently to its targets, creating an irreversible sink which will allow it to avoid efflux by MDR pumps of Gram negative species which is the main obstacle in developing broad-spectrum antibiotics. We will use E. coli, including the O157:H7 enterohemorrhagic strain that is a Category B waterborne agent in screen development. The process will include test strain construction- screening at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases facility in Boston and at an NIH Screening Center, and validation of hits to indicate their prodrug nature. This research will lead to the development of new antibiotics with a broad spectrum of action that will be effective against both bioweapons agents such as Y. pestis, F. tularensis and B. anthracis, and conventional pathogens. The uniquely novel property of these compounds will be their ability to completely sterilize an infection, preventing relapse of a disease and enabling effective treatment of immunodeficient individuals, including the elderly and the very young. [unreadable] [unreadable] [unreadable]